Simple Two-step Purification Procedure Research Articles

A highly efficient method for the production and purification of recombinant human CXCL8.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.

A hemorrhagic metalloprotease of Egyptian Cerastes vipera venom: Biochemical and immunological properties.

A hemorrhagic metalloprotease (CVHT1) was isolated from Cerastes vipera (CV) viper venom and characterized in a set of biochemical and immunological assays. A simple two-step purification procedure included gel filtration and ion-exchange increase the purity of enzyme 39-fold with specific activity of 20,200 Umg-1 compared to 520 Umg-1 for crude venom. CVHT1 is a dimer enzyme with two subunits of ~60 kDa. The LC-MS/MS analysis of CVHT1 revealed that the identified peptides show high homology to other P-III snake venom zinc-metalloproteases. The activity of CVHT1 showed stability at pH (6.5-8.5) and temperature (30-60 °C) with optima at pH 8.5 and 60 °C. Activators for CVHT1 included Mg+2, Zn+2, Ca+2, K+, Ba+2 and Na+, while the full inhibition was given by other tested ions, SH-group reagents and metalloproteinase inhibitors. The CVHT1 potentially digested gelatin, fibrinogen, fibronectin and inhibited the platelet aggregation. The hemorrhagic and proteolytic activities of medically important Egyptian viper venoms were highly cross-neutralized by anti-CVHT1. The anti-CVHT1 increased the survival time of mice injected with lethal dose of CV venom to 23 ± 2.5 h compared to the mice injected with venom alone 0.52 ± 0. 05 h. This study could be useful for production of safer and more efficient therapeutic anti-venom.

Isolation of Plasmodesmata.

Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology. Here we outline a simple two-step purification procedure that allows isolation of PD-derived membranes from Arabidopsis suspension cells. The method involves isolation of purified cell wall fragments containing intact PD which is followed by enzymatic degradation of the cell wall to release the PD. This membrane-rich fraction can be subjected to protein and lipid extraction for molecular characterization of PD components. The first step of this procedure involves the isolation of cell wall fragments containing intact PD, free from contamination from other cellular compartments. Purified PD membranes are then released from the cell wall matrix by enzymatic degradation. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

High expression of recombinant human catalase and its immunomodulatory effects on H1N1 influenza virus infection

Catalase is a key antioxidant enzyme and has been implicated in many pathophysiological processes of human diseases. In a previous study, we developed a yeast-based expression system for recombinant human catalase (rhCAT), and proved its therapeutic effects for treating H1N1 virus-induced pneumonia. However, the preparation of rhCAT was insufficient for further research applications. Here, we describe a much more convenient construction strategy for rhCAT based on the Pichia pastoris GS115 strain in a 14L bioreactor. Quantitative-PCR, Western blotting, and activity assay were used to demonstrate the stable and efficient high-level expression (1500U/mL; 77% recovery) achieved by this newly developed simple two-step purification procedure. The rhCAT synthesized by this new procedure was applied to an H1N1-infected mouse model (doses of 50 and 100kU/mice/day) to assess its capabilities for inducing immunomodulatory effects. The results showed that the rhCAT could restore the impaired phagocytosis, alleviate the induced reductions in spleen and thymus organ weights, and markedly reduce the lung tissue viral load, all in a dose-dependent manner. Thus, this study provides not only a simple method for large-scale preparation of active rhCAT, but also in vivo evidence of the recombinant protein's immunomodulatory activity which may have clinical applications in treating H1N1 or other viral infections.

Characterization of a Novel N-Acetylneuraminate Lyase from Staphylococcus carnosus TM300 and Its Application to N-Acetylneuraminic Acid Production

The possibility of incorporating N-acetylneuraminic acid (Neu5Ac) in infant formulas and other functional foods has opened up the need to synthesize N-acetylneuraminic acid using N-acetylneuraminate lyases (NALs) by reversible aldol condensation of pyruvate and N-acetyl-d-mannosamine. Until now, NALs have been cloned from pathogenic microorganisms; however, this Article describes the expression and characterization of an N-acetylneuraminate lyase from the Staphylococcus carnosus TM300, a GRAS microorganism used in fermented meat. ScNAL showed a high level of expression in E. coli (403 mg L(-1) culture). This, combined with its simple two-step purification procedure, the highest recovery described to date (86%), its kinetic parameters, which are in the same order of magnitude as best reported NALs, and its optimum pH and temperature, make ScNAL a promising and cheap biocatalyst. To confirm its biotechnological potential, the Neu5Ac was synthesized in 3 h in simple industrial working conditions with a high degree of conversion (94%).

Induction, purification and partial characterization of vitellogenin in an Indian major carpCatla catla(Ham.)

Vitellogenin was purified from the serum of 17-β estradiol (E2)-induced juvenile Catla catla using a simple two-step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS-PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone-induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.

A simple two-step purification procedure for the iC3b binding collectin conglutinin

Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81μg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (Kd=2.3−3.2nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.

Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties

The mature lipase of the fungus Rhizopus oryzae (ROL) was functionally expressed and secreted in the methylotrophic yeast Pichia pastoris. In a batch cultivation, where methanol feeding was linked to the dissolved oxygen content in the cultivation solution, a lipase activity of 500 000 units per liter (60 mg active lipase per liter) of culture was achieved after initial glycerol feeding of the culture. Recombinant ROL lipase was purified to homogeneity by a simple two-step purification procedure and had a specific activity of 8571 U mg −1 (triolein, 30°C, pH 8.1) which is comparable with the purified native enzyme. The properties of the recombinant lipase were similar to those reported both for the native lipase and for the enzyme expressed in Escherichia coli and refolded from inactive inclusion bodies.

Simple two-step procedure for purification of cloned small sialidase from unclarified E. coli feedstocks

An efficient process for the purification of cloned small sialidase (pCPN-1, small nanH cloned from Clostridium perfringens) from unclarified E. coli feedstocks has been developed. In contrast to conventional purification processes, expanded bed adsorption (EBA) technique allows clarification, product recovery, and concentration to be combined into a single step operation. The cloned NanH was collected from both intra- and extracellular fractions, and purified by expanded bed ion-exchange chromatography followed by affinity chromatography. The EBA technique using STREAMLINE DEAE was shown to be successful in purifying NanH with a purification factor of 12 and yield of 93%. Successive purification of the concentrated NanH purified by an EBA column was achieved by affinity chromatography. The elution of adsorbed NanH was performed with 0.1 m NaHCO 3 buffer pH 9.0 and the pH of the eluted NanH was adjusted to 7.0. An overall yield of 88% was obtained after the affinity chromatographic step. The enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis analysis. The molecular weight was measured as 43 KDa for cloned NanH. The procedure described is suitable for large-scale purification of cloned small NanH from unclarified E. coli feedstocks.

Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system

The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction.

Quantification, immunoaffinity purification and sequence analysis of a pigment-dispersing hormone of the shore crab, Carcinus maenas (L.)

1. 1. A sensitive sandwich-ELISA for pigment-dispersing hormone (PDH) with a detection limit of approximately 5 fmol per well has been developed using primary IgG and secondary biotinylated IgG fractions from an antiserum against β-PDH and streptavidin-peroxidase conjugate for detection. 2. 2. ELISA determinations in different parts of the central nervous system of the shore crab Carcinus maenas revealed maximum levels of 15.1 pmol immunoreactive PDH in the two eyestalk ganglia of one crab, 3.6 pmol in the sinus glands, 2.8 pmol in the brain and 0.8 pmol in the thoracic ganglia. 3. 3. Carcinus PDH was purified from whole eyestalk ganglia and sinus glands by use of a simple two-step purification procedure consisting of immunoaffinity-prepurification on an anti-PDH IgG-protein A-Sepharose column and HPLC. 4. 4. Automated gas-phase sequencing of the purified peptide and FAB-mass spectroscopy unambiguously revealed the sequence of Carcinus PDH as NSELINSILGLPKVMNDAamide ( M r 1927.2 Da), which is identical to the β-PDH of other brachyuran crustaceans.

Purine Nucleoside Phosphorylase: Purification Using an Ether-Linked Formycin B/Sepharose 6B Resin with Unusual Properties

Formycin B [9-deazainosine] was reacted with epoxy-activated Sepharose 6B to form an affinity resin for purine nucleoside phosphorylase (PNPase). This resin had a large capacity (7,600 units/ml) for the enzyme from Escherichia coli. Enzyme retention was dependent on high ionic strength. Although this property is reminiscent of hydrophobic interaction chromatography, analogous resins prepared with pseudouridine or monoethanolamine instead of with formycin B, did not retain the enzyme even at high ionic strength. Furthermore, hypoxanthine facilitatted elution of the enzyme from the resin. It appeared, therefore, that the enzyme was not bound simply by hydrophobic interactions. A simple two-step purification procedure for PNPase from Escherichia coli was devised using this resin. Overall recovery was 50%, and purity of the final preparation was greater than 95%. This resin was also useful in the purification of PNPase from human erythrocytes. The ether linkage between formycin B and Sepharose 6B, together with the carbon-to-carbon linkage between the pentose and heterocyclic moieties of formycin B, provided stability to both chemical and enzymatic degradation. After 5 years of use and exposure to a variety of biological preparations, the resin showed no detectable decrease in its ability to bind PNPase.

Isolation of trypsin isozymes from the mosquito Aedes aegypti (L.)

Trypsin has been isolated from midgut homogenates of blood-fed females of Aedes aegypti by a simple two-step purification procedure: ion-exchange chromatography followed by affinity chromatography. The resulting mosquito trypsin contains a number of isozymes, among which 5 major SDS-PAGE bands are recognized with molecular weights of 26.7, 28.5, 29.7, 31.0 and 32.0 kdaltons, as are some minor bands above and below this range. The isozymic pattern is comparable to that in crude homogenates. Isoelectric focussing of purified trypsin however, revealed over 20 tryptic isozymes, demonstrating that several isozymes segregate into subforms. A high correlation between TAME-active fractions and their DFP equivalent was demonstrated by using 3H-labelled DFP as a marker for trypsin on native acrylamide gels. The purification factor and the specific activities are discussed with respect to the unusual amounts of protein of dietary origin present in the midgut homogenates. Interference of blood-borne coagulation factors of a tryptic nature is unlikely.

Interaction between heparin and human pregnancy-associated plasma protein A (PAPP-A): A simple purification procedure

Human pregnancy-associated plasma protein A (PAPP-A) binds to heparin-Sepharose. This affinity chromatography preceded by molecular sieve chromatography provides a simple two-step purification procedure of PAPP-A from late pregnancy plasma. One hundred percent of the applied PAPP-A was recovered, with more than 40% being electrophoretically homogenous after the two procedures. The remaining PAPP-A could be purified by negative affinity chromatography on anti-total human serum immobilized on agarose.

Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties.

A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively.